Detection of staphylococcus aureus and identification of methicillin-resistant staphylococcus aureus

ABSTRACT

Aspects of the present invention relate to methods and compositions for the detection and/or quantification of  S. aureus  from a sample, as well as methods and compositions useful for the detection and/or quantification of  S. aureus  and MRSA in a single assay. Embodiments include nucleic acids that hybridize to  S. aureus -specific nuc sequences and MREJ sequences.

RELATED APPLICATIONS

The present application is a continuation of an claims priority to U.S.patent application Ser. No. 11/959,337, filed Dec. 18, 2007, to Jean etal., entitled “DETECTION OF STAPHYLOCOCCUS AUREUS AND IDENTIFICATION OFMETHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS,” which claims priority toU.S. Provisional Application Ser. No. 60/870,823, filed on Dec. 19,2006, to Jean et al. entitled “DETECTION OF STAPHYLOCOCCUS AUREUS ANDIDENTIFICATION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS,” each ofwhich are hereby expressly incorporated by reference in theirentireties.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledGENOM072C2.TXT, created Aug. 26, 2013, which is 129 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Members of the genus Staphylococcus are major human pathogens, causing awide variety of hospital and community acquired infections worldwide.The coagulase-positive species Staphylococcus aureus is well documentedas a human opportunistic pathogen (Murray et al. Eds, 2003, Manual ofClinical Microbiology, 8th Ed., ASM Press, Washington, D.C.). Nosocomialinfections caused by S. aureus are a major cause of morbidity andmortality. Some of the most common infections caused by S. aureusinvolve the skin, and they include furuncles or boils, cellulitis,impetigo, and postoperative wound infections at various sites. Some ofthe more serious infections produced by S. aureus are bacteremia,pneumonia, osteomyelitis, acute endocarditis, myocarditis, pericarditis,cerebritis, meningitis, scalded skin syndrome, and various abcesses.Food poisoning mediated by staphylococcal enterotoxins is anotherimportant syndrome associated with S. aureus. Toxic shock syndrome, acommunity-acquired disease, has also been attributed to infection orcolonization with toxigenic S. aureus.

Coagulase-negative Staphylococci had been regarded as harmless skincommensals prior to the 1970s, however, they are now recognized asimportant causes of human infections (Kloos, et al. (2004) Clin.Microbiol. Rev. 7:117-140). In addition to being among the mostfrequently isolated bacteria in clinical microbiology laboratories,coagulase-negative Staphylococci serve as reservoirs of antimicrobialresistance determinants (Bastos, et al. (1999) Eur. J. Clin. Microbiol.Infect. Dis. 18:393-398). As such, it is important to characterize anddistinguish S. aureus strains from other, coagulase-negativeStaphylococci.

S. aureus strains produce an extracellular thermostable nuclease(thermostable TNase) with a frequency similar to that at which theyproduce coagulase. The sequence of the gene encoding TNase, nuc, wasfirst disclosed in 1985 (Kovacevi et al. (1985), J. Bact. 162:521-528).TNase is a 17 kDa protein that degrades both RNA and DNA at temperaturesup to 100° C. TNase activity is not specific for S. aureus, however, S.aureus species-specific sequences exist. See, e.g., Brackstad, et al.(1992), J. Clin. Microbiol. 30:1654-1660; Zhang, et al. (2004), J. Clin.Microbiol. 42:4947-4955; Chesneau, et al. (1993) Mol. Cell. Probes7:301-310, Wilson, et al. (1991) Appl. Environ. Microbiol. 57:1793-1798;Poulsen et al., (2003) J. Antimicrob. Chemo. 51:419-421, Costa et al.,(2005), Diag. Microbiol. and Infect. Dis, 51: 13-17, Shittu et al.,(2006), Diagn Microbiol Infect Dis. 2006 Jul 17. To date, none of the S.aureus-specific nuc sequences have been proven to be clinically usefulby way of a large specificity study. Therefore, there exists a need foroligonucleotides that have been proven to be both highly specific andsensitive, which are useful in rapid detection and identification of S.aureus from clinical samples.

Both S. aureus and coagulase-negative Staphylococci have a remarkableability to accumulate additional antibiotic resistant determinants,resulting in the formation of multidrug-resistant strains. Thisresistance limits therapeutic options for treatment and substantiallyincreases patient morbidity and mortality. Methicillin-resistant S.aureus (MRSA) emerged in the 1980s as a major clinical and epidemiologicproblem in hospitals (Oliveira et al., (2002) Lancet Infect Dis.2:180-189). MRSA are resistant to all β-lactams including penicillins,cephalosporins, carbapenems, and monobactams, which are the mostcommonly used antibiotics to cure S. aureus infections. MRSA infectionscan only be treated with more toxic and more costly antibiotics, whichare normally used as the last line of defense. Since MRSA can spreadeasily from patient to patient via personnel, hospitals over the worldare confronted with the problem to control MRSA.

Methicillin resistance in S. aureus is unique in that it is due toacquisition of DNA from other coagulase-negative staphylococci (CNS),coding for a surnumerary β-lactam-resistant penicillin-binding protein(PBP), which takes over the biosynthetic functions of the normal PBPswhen the cell is exposed to β-lactam antibiotics. S. aureus normallycontains four PBPs, of which PBPs 1, 2 and 3 are essential. Thelow-affinity PBP in MRSA, termed PBP 2a (or PBP2′), is encoded by thechoromosomal mecA gene and functions as a β-lactam-resistanttranspeptidase. The mecA gene is absent from methicillin-sensitive S.aureus but is widely distributed among other species of staphylococciand is highly conserved (Ubukata et al., (1990) Antimicrob. AgentsChemother. 34:170-172).

Nucleotide sequence determination of the DNA region surrounding the mecAgene from S. aureus strain N315 (isolated in Japan in 1982), led to thediscovery that the mecA gene is carried by a novel genetic element,designated staphylococcal cassette chromosome mec (SCCmec), which isinserted into the chromosome. SCCmec is a mobile genetic elementcharacterized by the presence of terminal inverted and direct repeats, aset of site-specific recombinase genes (ccrA and ccrB), and the mecAgene complex (Ito et al., (1999) Antimicrob. Agents Chemother.43:1449-1458; Katayama et al., (2000) Antimicrob. Agents Chemother.44:1549-1555). SCCmec is precisely excised from the chromosome of S.aureus strain N315 and integrates into a specific S. aureus chromosomalsite in the same orientation through recombinases encoded by the ccrAand ccrB genes. Cloning and sequence analysis of the DNA surrounding themecA gene from MRSA strains NCTC 10442 (the first MRSA strain isolatedin England in 1961) and 85/2082 (a strain from New Zealand isolated in1985) led to the discovery of two novel genetic elements that sharedsimilar structural features of SCCmec. The three SCCmec have beendesignated type I (NCTC 10442), type II (N315) and type III (85/2082)based on the year of isolation of the strains (Ito et al., (2001)Antimicrob. Agents Chemother. 45:1323-1336). Hiramatsu et al. have foundthat the SCCmec DNAs are integrated at a specific site in the chromosomeof methicillin-sensitive S. aureus (MSSA). The nucleotide sequence ofthe regions surrounding the left and right boundaries of SCCmec DNA(i.e. attL and attR, respectively), as well as those of the regionsaround the SCCmec DNA integration site (i.e. attBscc which is thebacterial chromosome attachment site for SCCmec DNA), were analyzed.Sequence analysis of the attL, attR and attBscc sites revealed thatattBscc is located at the 3′ end of a novel open reading frame (ORF),orfX. orfX encodes a putative 159-amino acid polypeptide that exhibitssequence homology with some previously identified polypeptides ofunknown function (Ito et al., (1999) Antimicrob. Agents Chemother.43:1449-1458). Two new types of SCCmec, designated type IV and type Vwere recently described (Ma et al., (2002) Antimicrob. Agents Chemother.46:1147-1152, Ito et al., (2004) Antimicrob Agents Chemother.48:2637-2651, Oliveira et al., (2001) Microb. Drug Resist. 7:349-360).Oliveira et al. also recently reported the existence of SCCmec type VI.Oliveira et al., (2006), Antimicrob Agents Chemother. 50:3457-3459. Thesequence of the right extremity of some Staphylococcus strainsclassified as SCCmec type IV has been determined. See, Ma et al., (2002)Antimicrob. Agents Chemother. 46:1147-1152; Ito et al., (2001)Antimicrob. Agents Chemother. 45:1323-1336; Oliveira et al., (2001)Microb. Drug Resist. 7:349-360. Sequences from S. aureus strains CA05and 8/6-3P, classified as SCCmec type IV, were nearly identical over2000 nucleotides to that of type II SCCmec of S. aureus strain N315 (Maet al., (2002) Antimicrob. Agents Chemother. 46:1147-1152; Ito et al.,(2001) Antimicrob. Agents Chemother. 45:1323-1336).

Methods to detect and identify MRSA based on the detection of the mecAgene and S. aureus-specific chromosomal sequences have been described.See, Schuenck et al., Res. Microbiol., (2006), in press, Shittu et al.,(2006), Diagn Microbiol Infect Dis. July 17, Grisold et al., (2006),Methods Mol. Biol. 345: 79-89, Costa et al., (2005), Diag. Microbiol.and Infect. Dis, 51: 13-17, Mc Donald et al., (2005), J. Clin.Microbiol., 43: 6147-6149, Zhang et al., (2005), J. Clin. Microbiol. 43:5026-5033, Hagen et al. (2005), Int J Med Microbiol. 295:77-86, Maes, etal. (2002) J. Clin. Microbiol. 40:1514-1517, Saito et al., (1995) J.Clin. Microbiol. 33:2498-2500; Ubukata et al., (1992) J. Clin.Microbiol. 30:1728-1733; Murakami et al., (1991) J. Clin. Microbiol.29:2240-2244; Hiramatsu et al., (1992) Microbiol. Immunol. 36:445-453).Furthermore, Levi and Towner (2003), J. Clin. Microbiol., 41:3890-3892and Poulsen et al. (2003), J Antimicrob Chemother., 51:419-421 describedetection of methicillin resistance in coagulase-negative Staphylococciand in S. aureus using the EVIGENE™ MRSA Detection kit.

However, because the mecA gene is widely distributed in both S. aureusand coagulase-negative staphylococci, each of the methods describedabove are incapable of discriminating between samples containing bothmethicillin-sensitive S. aureus (“MSSA”) and methicillin-resistantcoagulase-negative staphylococci, and samples that contain only MRSA orthat have both methicillin-sensitive S. aureus and MRSA.

To address this problem, Hiramatsu et al. developed a PCR-based assayspecific for MRSA that utilizes primers that hybridize to the rightextremities of DNA of SCCmec types I-III in combination with primersspecific to the S. aureus chromosome, which corresponds to thenucleotide sequence on the right side of the SCCmec integration site.(U.S. Pat. No. 6,156,507, hereinafter the “'507 patent”). More recently,Zhang et al., (2005), J. Clin. Microbiol. 43: 5026-5033, described amultiplex assay for subtyping SCCmec types I to V MRSA. Nucleotidesequences surrounding the SCCmec integration site in otherstaphylococcal species (e.g., S. epidermidis and S. haemolyticus) aredifferent from those found in S. aureus, therefore multiplex PCR assaysthat utilize oligonucleotides that hybridize to the right extremities ofSCCmec and the S. aureus chromosome have the advantage of being specificfor the detection of MRSA.

The PCR assay described in the '507 patent also led to the developmentof “MREP typing” (mec right extremity polymorphism) of SCCmec DNA (Itoet al., (2001) Antimicrob. Agents Chemother. 45:1323-1336; Hiramatsu etal., (1996) J. Infect. Chemother. 2:117-129). The MREP typing methodtakes advantage of the fact that the nucleotide sequences of the threeMREP types differ at the right extremity of SCCmec DNAs adjacent to theintegration site among the three types of SCCmec. Compared to type I,type III has a unique nucleotide sequence while type II has an insertionof 102 nucleotides to the right terminus of SCCmec. The MREP typingmethod described by Hiramatsu et al. uses the following nomenclature:SCCmec type I is MREP type i, SCCmec type II is MREP type ii, and SCCmectype III is MREP type iii. Hiramatsu later revised this nomenclature inview of the publication of the sequences of the genomes of strains N315and Mu50, since the sequences revealed that SCCmec elements are locateddownstream of orfX. Consequently, MREP can now be referred to as MLEP(mec left extremity polymorphism) (Chongtrakool et al., (2006),Antimicrob. Agents Chemother. 50:1001-1012).

Recently, Chongtrakool et al. proposed replacing the SCCmec nomenclaturewith new nomenclature. Chongtrakool et al., (2006), Antimicrob. AgentsChemother. 50:1001-1012. Chongtrakool et al.'s proposed nomenclature isbased on the structure of SCCmec elements and has three features. Thefirst feature is a description of the SCC type and is defined by ccrtype and mec class. The second feature is the description of the Jregions (junkyard regions), which are part of the SCCmec element,located between and around the mec and ccr complexes. The third featureis the enumeration which allows the numbering of ccr type and J regionsaccording to their time of identification.

As stated above, SCCmec types II and IV have the same nucleotidesequence to the right extremity. Consequently, the MREP (or MLEPaccording to recent revision) typing method described above cannotdifferentiate the SCCmec type IV described by Hiramatsu et al. (Ma etal., (2002) Antimicrob. Agents Chemother. 46:1147-1152) from SCCmec typeII).

We recently described DNA sequences and regions in MRSA named MREJ. PCTApplication No. PCT/CA02/00824. The phrase MREJ refers to the mec rightextremity junction <<mec right extremity junction>>. MREJ's areapproximately 1 kilobase (kb) in length and include sequences from theSCCmec right extremity as well as bacterial chromosomal DNA to the rightof the SCCmec integration site. Importantly, MREJ sequences provideadvantages over MREP/MLEP sequences in classifying MRSA in thatMREJ/MLEJ sequences enable the differentiation between strainsclassified as SCCmec type II and SCCmec type IV. As discussed in PCTApplication No. PCT/CA02/00824, the strains that Hiramatsu classified asMREP types i-iii fall under MREJ types i-iii according to the MREJtyping system. We recently identified novel MREJ types iv-xx, anddeveloped nucleic acid assays with improved ubiquity capable ofdetection and identification of MRSA of MREJ types i-xx. (Huletsky etal., 2004, J Clin. Microbiol. 42:1875-1884, International PatentApplication PCT/CA02/00824, U.S. patent application Ser. No.11/248,438). Based on the revision of MREP to MLEP, one can understandthat previously called MREJ types could now be reclassified as MLEJ (mecleft extremity junction). The skilled artisan will appreciate that anyS. aureus and MRSA classification system is contemplated in the methodsdisclosed herein, as sequences can specifically detect S. aureus andidentify those which are resistant to methicillin.

Maes et al. describe a PCR assay to discriminate S. aureus fromcoagulase negative Staphylococci and to determine methicillin resistancein blood cultures (Maes, et al. (2002) J. Clin. Microbiol.40:1514-1517). The assay described in Maes et al. cannot distinguishMRSA from methicillin-resistant coagulase-negative Staphylococci.

Poulsen et al. describe detection of methicillin resistance incoagulase-negative Staphylococci and in S. aureus using the EVIGENE™MRSA Detection kit. The assay described in Poulsen et al. cannotdiscriminate between a sample that has both methicillin-sensitive S.aureus and methicillin-resistant coagulase-negative staphylococci, and asample that contains only MRSA or that has both methicillin-sensitive S.aureus and MRSA.

Accordingly, there remains a need for a rapid assay to detect andidentify both MRSA and methicillin-sensitive S. aureus in the samereaction and to be able to distinguish S. aureus from coagulase-negativeStaphylococci in the same reaction.

SUMMARY OF THE INVENTION

Disclosed herein are methods and compositions for specifically detectingthe presence of a Staphylococcus aureus (S. aureus) strain and detectingthe presence of a methicillin-resistant S. aureus (MRSA) strain from aclinical sample in a single assay. Also provided herein are methods andcompositions for the specific detection of S. aureus from a sample.

Some embodiments relate to methods of detecting S. aureus andidentifying the presence of MRSA from a sample that includes nucleicacids. In some embodiments, the sample can be contacted with at leastone primer and or probe of at least 10 nucleotides that anneals understringent conditions a S. aureus-specific sequence of the nuc gene, andat least one primer and/or probe specific for a MRSA strain. S aureusstrains are rendered methicillin-resistant due to the presence of anSCCmec cassette containing a mecA gene that is inserted in bacterialnucleic acids. The insertion of the SCCmec cassette can generate apolymorphic right extremity junction (MREJ). The MRSA-specific primer(s)and/or probe(s) can anneal under stringent conditions to polymorphicMREJ nucleic acids, including, for example, MREJ types i to xx. S.aureus-specific and MRSA-specific primers anneal under conditions of,for example, 4 mM MgCl₂, 100 mM Tris (pH 8.3), 10 mM KCl, and 5 mM(NH₄)₂SO₄ at 59° C. The presence and/or amount of annealed probe(s), oramplification products produced through annealing of the primers to thenucleic acids, can be used as an indication of the presence and/oramount of S. aureus (MSSA and MRSA) and MRSA in the sample.

The at least one primer specific for a S. aureus strain can anneal understringent conditions to the SEQ ID NO: 200, the complement thereof orany sequence which differs from SEQ ID NO: 200 by 1 to 20 nucleotides.

In some embodiments, the at least one primer and/or probe that annealsunder stringent conditions to the S. aureus specific nuc sequencehybridizes under stringent conditions to one of the following SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or the complement thereof.Preferably, the at least one primer and/or probe that anneals understringent conditions to the S. aureus specific nuc sequence comprises,consists essentially of, or consists of one of the following SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In preferred embodiments, the S. aureus-specific primer(s) and/orprobe(s) are at least 10 nucleotides in length, and anneal understringent conditions to the nucleic acid of any one of SEQ ID NOs: 1 to12 or the complement thereof.

In still more preferred embodiments, the sample is also contacted with aprobe that anneals under stringent conditions to the nucleic acid of anyone of SEQ ID NOs: 9, 10, 11, or 12, or the complement thereof. In someembodiments, the probe is a molecular beacon probe. Preferably, theprobe comprises, consists essentially of, or consists of the sequence ofSEQ ID NOs: 9, 10, 11, or 12.

In some embodiments, the method also includes adding internal controlDNA to the sample, and at least one primer and/or probe that annealsunder stringent conditions to the internal control DNA. For example, insome embodiments, the Internal Control can be a linearized 4.23 kbplasmid purified from E. coli. The internal control can be used tomonitor the presence of inhibitory substances coming from a specimen.

The at least one primer specific for an MRSA strain can anneal understringent conditions to the MREJ sequences of types i to xx, as definedin any one of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, and 88, the complement thereof orany sequence which differs from SEQ ID NOs 14 to 88 by 1 to 20nucleotides.

In preferred embodiments, S. aureus-specific and MRSA-specific primersand/or probes are chosen to anneal to the sample nucleic acids under thesame annealing conditions. In more preferred embodiments, the primer(s)and/or probe(s) are placed altogether in the same physical enclosure.

In preferred embodiments, the MRSA-specific primer(s) and/or probe(s)are at least 10 nucleotides in length, and anneal under stringentconditions to the nucleic acid of any one of SEQ ID NOs: 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 201 (types i-ix) 182, 183, 184, 195, 186, 187, 188, 189,190, 191, 193, 194, 195, 196, 197, 198 (types x-xx) or 199 or thecomplement thereof. Preferably, the MRSA-specific primer(s) and/orprobe(s) comprise, consist essentially of, or consist of the nucleicacid of any one of SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201(types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194,195, 196, 197, (types xi-xx) or 199. In some embodiments, MRSA-specificprimers also include an oligonucleotide that hybridizes under stringentcondition to orf22 of the S. aureus chromosome, wherein the primer canbe used in an amplification reaction with SEQ ID NO: 197 to detect MREJtype x. In more preferred embodiments, the MRSA-specific primer(s)and/or probe(s) anneal under stringent conditions to the nucleic acid ofany one of SEQ ID NOs: 99, 199, 144, 150, 155, and 163 or the complementthereof, such as a primer and/or probe that comprises, consistsessentially of, or consists of the nucleic acid of any one of SEQ IDNOs: 99, 199, 144, 150, 155, and 163. In still more preferredembodiments, the sample is also contacted with a probe that annealsunder stringent conditions to the nucleic acid of any one of SEQ ID NOs:126, 128, 130 and 131, or the complement thereof. In some embodiments,the probe is a molecular beacon probe. Preferably, the probe comprises,consists essentially of, or consists of the nucleic acid of any one ofSEQ ID NOs: 126, 128, 130 and 131.

In some embodiments, the sample and primer(s) and/or probe(s) describedabove are used in an amplification reaction, such as a PCR, LCR, NABSA,3SR, SDA, bDNA, TMA, CPT, SPA, NDSA, rolling circle amplification,anchored-strand displacement amplification, solid-phase (immobilized)rolling circle amplification, or Q beta replicase amplificationreaction.

Other aspects relate to the specific detection of an S. aureus strain ina sample that includes nucleic acids. At least one primer and/or probethat is specific for the nuc gene of S. aureus is provided. The primersand/or probe(s) include a nucleic acid that can anneal to at least 11consecutive nucleotides of any one of the nucleic acids of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or the complement thereof,under stringent conditions, such as 50 mM KCl, 10 mM Tris-HCl (pH 9.0),0.1% Triton X-100, 2.5 mM MgCl₂ at 59° C.; or 4 mM MgCl₂, 100 mM Tris(pH 8.3), 10 mM KCl, and 5 mM (NH₄)₂SO₄ at 59° C. The primer(s) and/orprobe(s) are allowed to anneal to the nucleic acids of the sample.Annealed primer(s) and/or probe(s) indicate the presence of an S. aureusstrain in the sample. The annealed primer(s) and/or probe(s) can bedetected, and the presence and/or amount of annealed probe(s), theamount of an amplification product produced through annealing of theprimers to the nucleic acids, indicates the presence and/or amount of S.aureus present in the sample.

In some embodiments, the sample and primer(s) and/or probe(s) describedabove are used in an amplification reaction, such as a PCR, LCR, NABSA,3SR, SDA, bDNA, TMA, CPT, SPA, NDSA, rolling circle amplification,anchored-strand displacement amplification, solid-phase (immobilized)rolling circle amplification, or Q beta replicase amplificationreaction.

In preferred embodiments, a primer pair including a first primer thatanneals under stringent conditions to SEQ ID NO:1 or the complementthereof (such as a primer that comprises, consists essentially of, orconsists of SEQ ID NO: 1), and a second primer that anneals understringent conditions to SEQ ID NO: 6 (such as a primer that comprises,consists essentially of, or consists of SEQ ID NO: 6), or the complementthereof, is allowed to anneal to the nucleic acids of the sample. Inmore preferred embodiments, a probe that anneals under stringentconditions to SEQ ID NO: 9 or 10 (such as a probe that comprises,consists essentially of, or consists of SEQ ID NO: 9 or 10), or thecomplement thereof, is also provided.

In other preferred embodiments, a primer pair including a first primerthat anneals under stringent conditions to SEQ ID NO: 3 (such as aprimer that comprises, consists essentially of, or consists of SEQ IDNO: 3)or the complement thereof, and a second primer that anneals understringent conditions to SEQ ID NO: 8 (such as a primer that comprises,consists essentially of, or consists of SEQ ID NO: 8) or the complementthereof, is allowed to anneal to the nucleic acids of the sample. Inmore preferred embodiments, a probe that anneals under stringentconditions to SEQ ID NO: 11 or 12, (such as a primer that comprises,consists essentially of, or consists of SEQ ID NO: 11 or 12) or thecomplement thereof, is also provided.

In still other preferred embodiments, the sample is contacted with atleast one primer pair, that includes a first primer and a second primerthat anneal under stringent conditions to the nucleic acid sequence ofat least one of the following pairs:

SEQ ID NOs: 1 and 5;

SEQ ID NOs: 1 and 6;

SEQ ID NOs: 2 and 5;

SEQ ID NOs: 2 and 6;

SEQ ID NOs: 3 and 7

SEQ ID NOs: 3 and 8;

SEQ ID NOs: 4 and 7; and

SEQ ID NOs: 4 and 8, or the complements thereof.

For example, in preferred embodiments, the sample is contacted with atleast one primer pair that includes a first primer and a second primerthat comprise, consist essentially of, or consist of the nucleic acidsequence of at least one of the following pairs:

SEQ ID NOs: 1 and 5;

SEQ ID NOs: 1 and 6;

SEQ ID NOs: 2 and 5;

SEQ ID NOs: 2 and 6;

SEQ ID NOs: 3 and 7

SEQ ID NOs: 3 and 8;

SEQ ID NOs: 4 and 7; and

SEQ ID NOs: 4 and 8.

In preferred embodiments, the sample is also contacted with at least oneprimer pair including a first primer and a second primer that annealunder stringent conditions to the nucleic acid sequence of at least oneof the following pairs:

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163, or the complement thereof.

For example, in some embodiments, the sample is contacted with at leastone primer pair including a first primer and a second primer thatcomprise, consist essentially of, or consist of at least one of thefollowing pairs:

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163.

In preferred embodiments, the sample is contacted with a plurality ofprimer pairs, wherein the primers anneal under stringent conditions tothe nucleic acid sequences of

SEQ ID NOs: 1 and 6

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163, or the complements thereof, such as primer pairsthat comprise, consist essentially of, or consist of the nucleic acidsequences of:

SEQ ID NOs: 1 and 6

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163.

In other preferred embodiments, the sample is contacted with a pluralityof primer pairs, wherein the primers anneal under stringent conditionsto the nucleic acid sequences of

SEQ ID NOs: 3 and 8

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163, or the complements thereof, such as primer pairsthat comprise, consist essentially of, or consist of the nucleic acidsequences of:

SEQ ID NOs: 3 and 8

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163.

Preferably, the sample is also contacted with at least one probe thatanneals under stringent conditions to the nucleic acid sequence of anyone of SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 and 131 or thecomplement thereof, such as a probe that comprises, consists essentiallyof, or consists of the nucleic acid sequence of any one of SEQ ID NOs:9, 10, 11, 12, 126, 128, 130 and 131.

Other aspects relate to oligonucleotides useful for the specificdetection of S. aureus. Some embodiments provide oligonucleotides whichanneal under stringent conditions with at least 11 consecutivenucleotides of the nucleic acid sequence of one of the following SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, such as nucleic acidsthat comprise, consist essentially of, or consist of one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

Yet other aspects relate to kits for detecting the presence of an S.aureus strain in a sample that includes nucleic acids. The kit caninclude at least one oligonucleotide that anneals under stringentconditions to one of the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or 12, or the complement thereof. For example, the kit caninclude at least one oligonucleotide that comprises, consistsessentially of, or consists of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12. In preferred embodiments, the kit also includes at leastone probe, wherein the probe can anneal to the nucleic acid sequence ofSEQ ID NO: 9, 10, 11 or 12, or the complement thereof, under stringentconditions. In preferred embodiments, the probe can comprise, consistessentially of, or consist of SEQ ID NO: 9, 10, 11 or 12.

In preferred embodiments, the kit also includes at least one primerspecific for an MRSA strain. S aureus strains are renderedmethicillin-resistant due to the presence of an SCCmec insert containinga mecA gene that is inserted in bacterial nucleic acids. The insertionof the SCCmec insert can generate a polymorphic right extremity junction(MREJ). The MRSA-specific primer(s) and/or probe(s) can anneal understringent conditions to polymorphic MREJ nucleic acids, including, forexample, MREJ types i to xx.

In preferred embodiments, the kit includes at least one MRSA-specificoligonucleotide that anneals under stringent conditions to one of thefollowing SEQ ID NOs: SEQ ID NOs:14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, and 88, or the complementthereof. For example, in some embodiments, a kit can contain at leastone MRSA-specific oligonucleotide that is at least 10 nucleotides inlength, and anneals under stringent conditions to the nucleic acidsequence of any one of the following SEQ ID NOs: 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 201 (types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190,191, 193, 194, 195, 196, 197, (types xi-xx) or 199 or the complementthereof. In some embodiments, the MRSA-specific oligonucleotides canalso include an oligonucleotide that hybridizes under stringentconditions to orf22 of the S. aureus chromosome, wherein theoligonucleotide can be used in an amplification reaction with SEQ ID NO:197 to detect MREJ type x. Preferably, the MRSA-specific oligonucleotidecan comprise, consist essentially of, or consist of any one of thefollowing SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 15, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201 (types i-ix)182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196,197, (types xi-xx) or 199.

In some embodiments, the kit contains a plurality of oligonucleotidesthat anneal under stringent conditions to SEQ ID NOs: 1, 6, 99, 144,150, 155, and 163. For example, in preferred embodiments, the kit cancontain a plurality of oligonucleotides that comprise, consistessentially of, or consist of SEQ ID NOs: 1, 6, 99, 144, 150, 155, and163. In some embodiments, the kit contains a plurality ofoligonucleotides that anneal under stringent conditions to SEQ ID NOs:3, 8, 99, 144, 150, 155, and 163, such as a plurality ofoligonucleotides that comprise, consist essentially of, or consist ofSEQ ID NOs: 3, 8, 99, 144, 150, 155, and 163. Preferably, the kit alsoincludes at least one probe that anneals under stringent conditions tothe following SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 or 131, such asat least one probe that comprises, consists essentially of, or consistsof SEQ ID NOs: 9, 10, 11, 12, 126, 128, 130 or 131.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show photographs of agarose gels showing the products ofPCR amplification reactions. The number of copies and source of templateDNA are indicated (15 cp=15 copies; 185 cp=185 copies) (S. aureus=MSSAstrain ATCC 25923; MRSA=MRSA strain ATCC 43300). Arrows indicate the PCRproduct sizes and primer dimers.

FIGS. 2A and 2B show a graphical depiction of PCR amplification curvesmeasured from reactions containing molecular beacon probes. Reactionscontained 0, 2.5, 5, 10, 15, or 20 copies of MSSA (FIG. 2A) or MRSA(FIG. 2B) template DNA, as well as 3000 copies of internal control DNA.Molecular beacon probes were added to each reaction and the fluorescenceof the reactions was measured. FAM labeled probes hybridize toMRSA-specific sequences, TET-labeled probes hybridize to internalcontrol DNA sequences, and Texas-Red-labeled probes hybridize to S.aureus-specific nuc sequences.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Methods and compositions disclosed herein relate to detection and/orquantification of S. aureus in a sample, and also relate to detectionand/or quantification of a Staphylococcus aureus (S. aureus) strain andidentification a methicillin-resistant S. aureus strain from a sample ina single assay. The embodiments disclosed herein are useful fordetection and/or quantification of S. aureus and MRSA from any type ofsample, such as any clinical sample, any environmental sample, anymicrobial culture, any microbial colony, any tissue, and any cell line.

Staphylococci are Gram-positive cocci. S. aureus can be distinguishedfrom other clinically relevant species of Staphylococcus by a positiveresult on the basis of their ability to clot blood plasma (the coagulasereaction) and their ability to form clumps in the presence offibrinogen. S. aureus, as some other staphylococci has the ability toproduce a thermostable nuclease (TNase), Becker et al., (2005), DiagnMicrobiol Infect Dis., 51:237-244, Brakstad et al, (1995), APMIS,103:219-224, Chesneau, et al. (1993) Mol. Cell. Probes 7:301-310.Nevertheless, some nucleotide sequences in the gene encoding thenuclease are specific of S. aureus strains (Costa et al., (2005), Diag.Microbiol. and Infect. Dis, 51: 13-17, Mc Donald et al., (2005), J.Clin. Microbiol., 43: 6147-6149, Zhang, et al. (2004), J. Clin.Microbiol. 42:4947-4955; Maes, et al. (2002) J. Clin. Microbiol.40:1514-1517).

Methods of Detecting S. aureus or S. aureus and MRSA

Some embodiments relate to methods of specifically detecting S. aureusin a sample. Disclosed herein are novel primers and/or probes (e.g., SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) that anneal to S.aureus-specific sequences of the nuc gene, exemplified by SEQ ID NO:200, which are useful to distinguish S. aureus from other Staphylococci,as well as other TNase-producing species of bacteria. In someembodiments, at least one primer and/or probe that anneals understringent conditions to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12, or the complement thereof is provided. For example, in someembodiments, the at least one primer and/or probe can comprise, consistessentially of, or consist of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12. The at least one primer is allowed to anneal to the nucleicacids of the sample, e.g., under standard PCR conditions or stringentconditions. The presence and/or amount of annealed probe(s) and/or theamount of an amplification product produced through annealing of theprimers to the nucleic acids, is detected, thereby indicating thepresence and/or amount of S. aureus present in the sample.

The term “consisting essentially of,” when used in reference to nucleicacid can refer to the specified nucleic acid sequences, and can includeany additional nucleotide that does not materially affect the basic andnovel characteristics of the specified sequence. The term “consistingessentially of” also can refer to variants that are substantiallysimilar to, and differ from a reference sequence in an inconsequentialway as judged by examination of the sequence. For example, nucleic acidsequences encoding the same amino acid sequence are substantiallysimilar despite differences in degenerate positions or modestdifferences in length or composition of any non-coding regions.

Primers and/or Probes and Nucleotides

As used herein, the terms “primer” and “probe” are not limited tooligonucleotides or nucleic acids, but rather encompass molecules thatare analogs of nucleotides, as well as nucleotides. Nucleotides andpolynucleotides, as used herein shall be generic topolydeoxyribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide which is an N- or C-glycoside of a purine or pyrimidinebase, and to other polymers containing nonnucleotidic backbones, forexample, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as NEUGENE™ polymers), and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA.

The terms nucleotide and polynucleotide include, for example,3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′→P5′ phosphoramidates,2′-O-alkyl-substituted RNA, double- and single-stranded DNA, as well asdouble- and single-stranded RNA, DNA:RNA hybrids, and hybrids betweenPNAs and DNA or RNA. The terms also include known types ofmodifications, for example, labels which are known in the art,methylation, “caps,” substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide or oligonucleotide.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only theknown purine and pyrimidine bases, but also other heterocyclic baseswhich have been modified. Such modifications include methylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Modified nucleosides or nucleotides will also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with a halogen, an aliphatic group, or are functionalized asethers, amines, or the like. Other modifications to nucleotides orpolynucleotides involve rearranging, appending, substituting for, orotherwise altering functional groups on the purine or pyrimidine basewhich form hydrogen bonds to a respective complementary pyrimidine orpurine. The resultant modified nucleotide or polynucleotide may form abase pair with other such modified nucleotidic units but not with A, T,C, G or U. For example, guanosine(2-amino-6-oxy-9-beta.-D-ribofuranosyl-purine) may be modified to formisoguanosine (2-oxy-6-amino-9-.beta.-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base which will no longereffectively form a standard base pair with cytosine. However,modification of cytosine(1-.beta.-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine(1-.beta.-D-ribofuranosyl-2-amino-4-oxy-pyrimidine) results in amodified nucleotide which will not effectively base pair with guanosinebut will form a base pair with isoguanosine. Isocytosine is availablefrom Sigma Chemical Co. (St. Louis, Mo.); isocytidine may be prepared bythe method described by Switzer et al. (1993) Biochemistry32:10489-10496 and references cited therein;2′-deoxy-5-methyl-isocytidine may be prepared by the method of Tor etal. (1993) J. Am. Chem. Soc. 115:4461-4467 and references cited therein;and isoguanine nucleotides may be prepared using the method described bySwitzer et al. (1993), supra, and Mantsch et al. (1993) Biochem.14:5593-5601, or by the method described U.S. Pat. No. 5,780,610 toCollins et al. The non-natural base pairs referred to as κ and π, may besynthesized by the method described in Piccirilli et al. (1990) Nature343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement(1-methylpyrazolo[4,3]-pyrimidine-5,7-(4H,6H)-dione. Other such modifiednucleotidic units which form unique base pairs have been described inLeach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al.,supra, or will be apparent to those of ordinary skill in the art.

Primers and/or probes are preferably between 10 and 45 nucleotides inlength. For example, the primers and or probes can be at least 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or morenucleotides in length. Primers and/or probes can be provided in anysuitable form, included bound to a solid support, liquid, andlyophilized, for example.

Annealing and Specific Binding

Binding or annealing of the primers and/or probes to target nucleic acidsequences is accomplished through hybridization. It will be appreciatedby one skilled in the art that specific hybridization is achieved byselecting sequences which are at least substantially complementary tothe target or reference nucleic acid sequence. This includesbase-pairing of the oligonucleotide target nucleic acid sequence overthe entire length of the oligonucleotide sequence. Such sequences can bereferred to as “fully complementary” with respect to each other. Wherean oligonucleotide is referred to as “substantially complementary” withrespect to a nucleic acid sequence herein, the two sequences can befully complementary, or they may form mismatches upon hybridization, butretain the ability to hybridize under stringent conditions or standardPCR conditions as discussed below.

A positive correlation exists between probe length and both theefficiency and accuracy with which a probe will anneal to a targetsequence. In particular, longer sequences have a higher meltingtemperature (T_(m)) than do shorter ones, and are less likely to berepeated within a given target sequence, thereby minimizing promiscuoushybridization.

As used herein, “T_(m)” and “melting temperature” are interchangeableterms which refer to the temperature at which 50% of a population ofdouble-stranded polynucleotide molecules becomes dissociated into singlestrands. Formulae for calculating the T_(m) of polynucleotides are wellknown in the art. For example, the T_(m) may be calculated by thefollowing equation: T_(m)=69.3+0.41×.(G+C)%−6−50/L, wherein L is thelength of the probe in nucleotides. The T_(m) of a hybrid polynucleotidemay also be estimated using a formula adopted from hybridization assaysin 1 M salt, and commonly used for calculating T_(m) for PCR primers:[(number of A+T)×2° C. +(number of G+C)×4° C]. See, e.g., C. R. Newtonet al. PCR, 2nd Ed., Springer-Verlag (New York: 1997), p. 24. Other moresophisticated computations exist in the art, which take structural aswell as sequence characteristics into account for the calculation ofT_(m). A calculated T_(m) is merely an estimate; the optimum temperatureis commonly determined empirically.

Primer or probe sequences with a high G+C content or that comprisepalindromic sequences tend to self-hybridize, as do their intendedtarget sites, since unimolecular, rather than bimolecular, hybridizationkinetics are generally favored in solution. However, it is alsoimportant to design a probe that contains sufficient numbers of G:Cnucleotide pairings since each G:C pair is bound by three hydrogenbonds, rather than the two that are found when A and T (or A and U)bases pair to bind the target sequence, and therefore forms a tighter,stronger bond. Preferred G+C content is about 50%.

Hybridization temperature varies inversely with probe annealingefficiency, as does the concentration of organic solvents, e.g.,formamide, which might be included in a hybridization mixture, whileincreases in salt concentration facilitate binding. Under stringentannealing conditions, longer hybridization probes, or synthesis primers,hybridize more efficiently than do shorter ones, which are sufficientunder more permissive conditions. Preferably, stringent hybridization isperformed in a suitable buffer under conditions that allow the referenceor target nucleic acid sequence to hybridize to the probes. Stringenthybridization conditions can vary (for example from salt concentrationsof less than about 1 M, more usually less than about 500 mM andpreferably less than about 200 mM) and hybridization temperatures canrange (for example, from as low as 0° C. to greater than 22° C., greaterthan about 30° C. and (most often) in excess of about 37° C. dependingupon the lengths and/or the nucleic acid composition of the probes.Stringent hybridization temperatures for PCR range from 40 and 75° C.,preferably between 45 and 70° C., depending on lengths and compositionsof primers. Longer fragments may require higher hybridizationtemperatures for specific hybridization. As several factors affect thestringency of hybridization, the combination of parameters is moreimportant than the absolute measure of a single factor. Accordingly, byway of example, the term “stringent hybridization conditions” may beidentified by those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995). For example, the term “stringent conditions”encompasses standard PCR conditions, as described below.

For a review of PCR technology, including standard PCR conditions,applied to clinical microbiology, see DNA Methods in ClinicalMicrobiology, Singleton P., published by Dordrecht; Boston: KluwerAcademic, (2000) Molecular Cloning to Genetic Engineering White, B.A.Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997) and“PCR Methods and Applications”, from 1991 to 1995 (Cold Spring HarborLaboratory Press). Non-limiting examples of “PCR conditions” include theconditions disclosed in the references cited herein, and also in theexamples below, such as, for example, 50 mM KCl, 10 mM Tris-HCl (pH9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, with an annealing temperature of72° C.; or 4 mM MgCl₂, 100 mM Tris, pH 8.3, 10 mM KCl, 5 mM (NH₄)₂SO₄,0.15 mg BSA, 4% Trehalose, with an annealing temperature of 59° C., or50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂,with an annealing temperature of 55° C.

As used herein, when used to describe primers and/or probes, the terms“specific” or “species-specific” refer to primers and/or probes whichhybridize or anneal under stringent conditions and/or standard PCRconditions to nucleic acids of a specified species or type (e.g. S.aureus or MRSA), and which do not substantially anneal or hybridizeunder the same conditions to unrelated nucleic acids, such as nucleicacids other than the specified species or MREJ type.

In a preferred embodiment, the probes or primers described hereinhybridize under stringent conditions to target sequences (e.g., S.aureus specific nuc sequences or MREJ sequences). In other preferredembodiments, the primers or probes described herein exhibit 100%complementarity over at least 10 to 45 nucleotides in length. Forexample, the primers and or probes exhibit complementarity over at least10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,or more nucleotides to the target sequence. In some embodiments, theprimers or probes exhibit 100% complementarity to the target sequenceover 10 to 45 consecutive nucleotides in all but at least 1 position(e.g., the primer and/or probe contains a mismatch), 2 positions, 3positions, 4 positions, 5 positions, 6 positions, 7 positions or more.

Probes or primers that include sequences that can hybridize as describedherein and that also include a portion that does not hybridize to thetarget sequence (e.g., a tag or a marker), are also contemplated. Forexample, in some embodiments, the primer and/or probe can contain adetectable moiety, such as a fluorescent moiety, or any other detectablemarker, such as those described below. In some embodiments, the primerand/or probe may contain nucleic acid or other molecular components thatfacilitate subsequent manipulations, such as polymerization reactions,or enzymatic reactions such as digestion with restriction endonucleases,and the like, or that couple the primer and/or probe to a solid support.

Amplification and Detection

In the methods described herein, detection of annealed primers and/orprobes can be direct or indirect. For example, probes can be annealed tothe sample being tested, and detected directly. On the other hand,primers can be annealed to the sample being tested, followed by anamplification step. The amplified products can be detected directly, orthrough detection of probes that anneal to the amplification products.

In preferred embodiments, an amplification and/or detection step followsthe annealing step. In other preferred embodiments, detection occursduring the annealing step. Any type of nucleic acid amplificationtechnology can be used in the methods described herein. Non-limitingexamples of amplification reactions that can be used in the methodsdescribed herein include but are not restricted to: polymerase chainreaction (PCR) (See, PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS,ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed.Innis, Academic Press, Inc., N.Y. (Innis)), ligase chain reaction (LCR)(See, Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077;Barringer (1990) Gene 89:117), nucleic acid sequence-based amplification(NASBA), self-sustained sequence replication (3SR) (See, Guatelli (1990)Proc. Natl. Acad. Sci. USA, 87:1874), strand displacement amplification(SDA), branched DNA signal amplification bDNA, transcription-mediatedamplification (TMA) (See, Kwoh (1989) Proc. Natl. Acad. Sci. USA86:1173), cycling probe technology (CPT), nested PCR, multiplex PCR,solid phase amplification (SPA), nuclease dependent signal amplification(NDSA), rolling circle amplification technology (RCA), Anchored stranddisplacement amplification, solid-phase (immobilized) rolling circleamplification, Q Beta replicase amplification and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario). Theseand other techniques are also described in Berger (1987) MethodsEnzymol. 152:307-316; Sambrook, Ausubel, Mullis (1987) U.S. Pat. Nos.4,683,195 and 4,683,202; Amheim (1990) C&EN 36-47; Lomell J. Clin.Chem., 35:1826 (1989); Van Brunt, Biotechnology, 8:291-294 (1990); Wu(1989) Gene 4:560; Sooknanan (1995) Biotechnology 13:563-564.

In preferred embodiments, PCR is used to amplify nucleic acids in thesample. During DNA amplification by PCR, two oligonucleotide primersbinding respectively to each strand of the heat-denatured target DNAfrom the microbial genome, are used to amplify exponentially in vitrothe target DNA. Successive thermal cycles allow denaturation of the DNA,annealing of the primers and synthesis of new targets at each cycle.(Persing et al, (1993), Diagnostic Molecular Microbiology: Principlesand Applications, American Society for Microbiology, Washington, D.C.).

The skilled artisan will appreciate that standard amplificationprotocols may be modified to improve nucleic acid amplificationefficiency, including modifications to the reaction mixture. (Ralser etal., (2006), Biochem Biophys Res Commun., 347:747-51, Kang et al.,(2005), J Biochem Biophys Methods. (2005), 64:147-51, Chakrabarti andSchutt, (2002), Biotechniques, 32:866-874; Al-Soud and Radstrom, (2000),J. Clin. Microbiol., 38:4463-4470; Al-Soud and Radstrom, 1998, Appl.Environ. Microbiol., 64:3748-3753; Wilson, 1997, Appl. Environ.Microbiol., 63:3741-3751). Such modifications of the amplificationreaction mixture include but are not limited to the use of variouspolymerases or the addition of nucleic acid amplification facilitatorssuch as betaine, BSA, sulfoxides, protein gp32, detergents, cations, andtetramethylamonium chloride.

Detection of amplified nucleic acids may include any real-time orpost-amplification technologies known to those skilled in the art.Classically, the detection of PCR amplification products is performed bystandard ethidium bromide-stained agarose gel electrophoresis, however,the skilled artisan will readily appreciate that other methods for thedetection of specific amplification products, which may be faster andmore practical for routine diagnosis, may be used, such as thosedescribed in co-pending patent application WO01/23604 A2. Amplicondetection may also be performed by solid support or liquid hybridizationusing species-specific internal DNA probes hybridizing to anamplification product. Such probes may be generated from any sequencefrom the MREJ or nuc nucleic acid sequences provided herein, anddesigned to specifically hybridize to DNA amplification productsproduced utilizing the methods disclosed herein. Alternatively,amplicons can be characterized by sequencing. See, e.g., co-pendingpatent application WO01/23604 A2 for examples of detection andsequencing methods.

Other non-limiting examples of nucleic acid detection technologies thatcan be used in the embodiments disclosed herein include, but are notlimited to the use of fluorescence resonance energy transfer(FRET)-based methods such as adjacent hybridization of probes (includingprobe-probe and probe-primer methods) (See, J. R. Lakowicz, “Principlesof Fluorescence Spectroscopy,” Kluwer Academic/Plenum Publishers, NewYork, 1999)., TaqMan probe technology (See, European Patent EP 0 543942), molecular beacon probe technology (See, Tyagi et al., (1996) Nat.Biotech. 14:303-308.), Scorpion probe technology (See, Thewell (2000),Nucl. Acids Res. 28:3752), nanoparticle probe technology (See,Elghanian, et al. (1997) Science 277:1078-1081.) and Amplifluor probetechnology (See, U.S. Pat. Nos. 5,866,366; 6,090,592; 6,117,635; and6,117,986).

In preferred embodiments, molecular beacons are used for detection ofthe target nucleic acids. Molecular beacons are single strandedoligonucleotides that, unless bound to target, exist in a hairpinconformation. The 5′ end of the oligonucleotide contains a fluorescentdye. A quencher dye is attached to the 3′ end of the oligonucleotide.When the beacon is not bound to target, the hairpin structure positionsthe fluorophore and quencher in close proximity, such that nofluorescence can be observed. Once the beacon hybridizes with target,however, the hairpin structure is disrupted, thereby separating thefluorophore and quencher and enabling detection of fluorescence. (See,Kramer FR., 1996, Nat Biotechnol 3:303-8.). Other detection methodsinclude target gene nucleic acids detection via immunological methods,solid phase hybridization methods on filters, chips or any other solidsupport. In these systems, the hybridization can be monitored by anysuitable method known to those skilled in the art, includingfluorescence, chemiluminescence, potentiometry, mass spectrometry,plasmon resonance, polarimetry, colorimetry, flow cytometry orscanometry. Nucleotide sequencing, including sequencing by dideoxytermination or sequencing by hybridization (e.g. sequencing using a DNAchip) represents another method to detect and characterize targetnucleic acids, such as nuc or MREJ nucleic acid sequences.

Methods

In preferred embodiments, methods to detect a S. aureus strain in asample include the step of providing a primer pair, with a first and asecond primer. The first and the second primer can anneal understringent conditions to at least one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12, or the complements thereof, such as primers thatcomprise, consist essentially of, or consist of SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12. In preferred embodiments, the primer pairscomprise first and second primers that anneal under stringent conditionsto SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ IDNOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4and 7; or SEQ ID NOs: 4 and 8, or the complements thereof. For example,in preferred embodiments, the primer pairs can comprise, consistessentially of, SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8;SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8. The sample can be contactedwith and allowed to anneal to the primer pair. Preferably, anamplification reaction (e.g., PCR) is performed with the annealed primerpair to amplify S. aureus-specific nuc sequences using the techniquesdescribed herein. Amplification products can then be detected using anyof the methods described herein.

In some embodiments, the primer pair includes a first primer thatanneals under stringent conditions to SEQ ID NO: 1 or the complementthereof, and a second primer that anneals under stringent conditions toSEQ ID NO: 6, or the complement thereof, such as a primer pair thatcomprises, consists essentially of, or consists of SEQ ID NO: 1 and SEQID NO: 6. In some embodiments, the primer pair is used to amplify nucsequences present in the sample. Optionally, a probe that anneals understringent conditions to SEQ ID NO: 9 or 10 or the complement thereof isalso provided, for example, a probe that comprises, consists essentiallyof, or consist of SEQ ID NO: 9 or 10. In some embodiments, the probe isa molecular beacon probe, and the resulting amplification product can bedetected by the probe.

In other preferred embodiments, a first primer that anneals understringent conditions to SEQ ID NO: 3 or the complement thereof and asecond primer that anneals under stringent conditions to SEQ ID NO: 8,or the complement thereof, are provided, such as a primer pair thatcomprises, consists essentially of, or consists of SEQ ID NO: 3 and SEQID NO: 8. In some embodiments, the primer pair is used to amplify nucsequences present in the sample. Optionally, a probe that anneals understringent conditions to SEQ ID NO: 11 or 12 or the complement thereof isalso provided for example, a probe that comprises, consists essentiallyof, or consist of SEQ ID NO: 11 or 12. In some embodiments, the probe isa molecular beacon probe.

Other aspects of the invention relate to methods and compositions fordetecting the presence of S. aureus strains and identifying MRSA strainsfrom a sample in a single assay or reaction. The term “single assay” or“single reaction” is intended to refer to the situation in which stepsto detect S. aureus and steps to detect MRSA are performedsimultaneously, or at substantially the same time, for example in thesame physical enclosure. The skilled artisan will appreciate, however,that steps to detect S. aureus and steps to detect MRSA can also beperformed sequentially. In preferred embodiments, S. aureus and MRSA aresimultaneously detected, for example in a multiplex PCR reaction.

Some embodiments involve the steps of contacting the sample with atleast one primer and/or probe that anneals under stringent conditions toa species-specific sequence of the nuc gene of S. aureus, and contactingthe sample with at least one primer and/or probe that anneals understringent conditions to a sequence that is specific to MREJ sequences ofMRSA strains.

The MRSA-specific primer(s) and/or probe(s) can anneal under stringentconditions to polymorphic MREJ nucleic acids, including, for example,MREJ types i to xx. The phrase MREJ refers to the mec right extremityjunction <<mec right extremity junction>>. MREJ's are approximately 1kilobase (kb) in length and include sequences from the SCCmec rightextremity as well as bacterial chromosomal DNA to the right of theSCCmec integration site (See, Huletsky et al., (2004) J. Clin.Microbiol., 42:1875-1884). Based on the determination of thewhole-genome sequences of strain N315 and Mu50, the nomenclature wasrecently reviewed because SCCmec elements are located downstream (andnot upstream) of orfX. Consequently, MREP (Mec Right ExtremityPolymorphism) is also referred to as MLEP (Mec Left ExtremityPolymorphism). By a similar token, MREJ types can be referred to as MLEJ(mec left extremity junction). (Chongtrakool et al., (2006), Antimicrob.Agents Chemother. 50:1001-1012). Nevertheless, any equivalent way toclassify S. aureus and namely MRSA strains will be under the scope ofthis patent, since sequences will be able to specifically detect S.aureus and to identify those which are resistant to methicillin.

Non-limiting examples MREJ type i to xx sequences are listed in SEQ IDNOs: 14-88. Accordingly, in some embodiments, in addition to at leastone S. aureus-specific nuc primer and/or probe, (e.g., anoligonucleotide that hybridizes under stringent conditions to one of thefollowing SEQ ID NO: 200, the complement thereof or any sequence whichdiffers from SEQ ID NO: 200 by 1 to 20 nucleotides, at least one primerand/or probe that specifically anneals under stringent conditions to atleast one MREJ sequence of MREJ types i-xx (e.g., SEQ ID NOs: 14-88) orthe complement thereof is provided. Exemplary primers and probes andcombinations of primers and probes useful for the detection of MRSA ofMREJ types i-xx are found in, for example, International PatentApplication PCT/CA02/00824, and in U.S. patent application Ser. No.11/248,438, hereby expressly incorporated by reference in theirentireties. For example, in some embodiments, the at least oneMRSA-specific primer and/or probe provided in the method is at least 10nucleotides in length, and can hybridize under stringent conditions toone of the following SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201(MREJ types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193,194, 195, 196, 197, (MREJ types xi-xx) or 199, or the complementthereof. For example, the MRSA-specific primers can comprise, consistessentially of, or consist of one of the following SEQ ID NOs: 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 15, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 201 (MREJ types i-ix) 182, 183, 184, 195, 186,187, 188, 189, 190, 191, 193, 194, 195, 196, 197, (MREJ types xi-xx) or199. nuc-specific primers and/or probes (e.g., comprising anoligonucleotide that hybridizes under stringent conditions to one of thefollowing SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or thecomplement thereof, such as oligonucleotides that comprise, consistessentially of, or consist of one of the following SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12) and MRSA (i.e., MREJ)-specific primersand/or probes (e.g., comprising an oligonucleotide that hybridizes understringent conditions to SEQ ID NOs: 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,15, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 201(types i-ix) 182, 183, 184, 195, 186, 187, 188, 189, 190, 191, 193, 194,195, 196, 197, (types xi-xx) or 199, or the complement thereof) areannealed to the nucleic acids of the sample, and the presence ofannealed primers and/or probes, or amplification products producedtherefrom, is detected, indicating the presence and/or amount of S.aureus as well as MRSA. For example, in some embodiments, the sample iscontacted with at least one primer pair comprising oligonucleotides thathybridize under stringent conditions to SEQ ID NOs: 92 and 82; 92 and83; 92 and 84; 104 and 86; 104 and 87; 104 and 88; 99 and 89; 99 and 199(for the detection of MREJ type i); SEQ ID NOs: 92 and 82; 92 and 129;92 and 130; 93 and 83; and 92 and 84; 99 and 89; 99 and 199 (for thedetection of MREJ type ii); SEQ ID NOs: 92 and 136; 92 and 137; 92 and138; 99 and 202; 99 and 144 (for the detection of MREJ type iii); SEQ IDNOs: 92 and 141; 99 and 105; 99 and 150 (for the detection of MREJ typeiv); SEQ ID NOs: 92 and 146; 99 and 196; 99 and 155 (for the detectionof MREJ type v); SEQ ID NOs: 92 and 152; 99 and 161; (for the detectionof MREJ type vi); SEQ ID NOs: 92 and 153; 92 and 154; 99 and 162; 99 and163 (for the detection of MREJ type vii); SEQ ID NOs: 92 and 162; 92 and163; 99 and 170 (for the detection of MREJ type viii); SEQ ID NOs: 92and 168; 99 and 177 (for the detection of MREJ type ix); SEQ ID NOs: 197and an oligonucleotide that hybridizes under stringent conditions toorf22 (for the detection of MREJ type x); SEQ ID NOs: 189 and 106; 189and 99; 189 and 190; 189 and 109 (for the detection of MREJ type xi);SEQ ID NOs: 194 and 106; 194 and 99; 104 and 191; 194 and 109 (for thedetection of MREJ type xii); SEQ ID NOs: 177 and 106; 177 and 99; 177and 190; and 177 and 109 (for the detection of MREJ type xiii); SEQ IDNOs: 177 and 106; 177 and 99; 177 and 193; 177 and 109 (for thedetection of MREJ type xiv); SEQ ID NOs: 184 and 106; 108 and 99; 184and 191; 184 and 191 (for the detection of MREJ type xv); SEQ ID NOs: 89and 109 (for the detection of MREJ type xvi); SEQ ID NOs: 185 and 106;185 and 99; 185 and 191; 185 and 109 (for the detection of MREJ typexvii); SEQ ID NOs: 186 and 106; 186 and 99; 186 and 193; 186 and 109(for the detection of MREJ type xviii); SEQ ID NOs: 187 and 106; 107 and99; 187 and 913; 187 and 109 (for the detection of MREJ type xix); SEQID NOs: 188 and 106; 188 and 99; 188 and 913; and 188 and 109 (for thedetection of MREJ type xx), or the complement thereof.

The most clinically relevant MRSA strains have MREJ types i, ii, iii,iv, v, and vii. Accordingly, preferred methods and compositions relateto the detection of S. aureus and MRSA of MREJ types i-v and vii in asample. At least one S. aureus-specific nuc-specific primer and/or probeis provided, and primers and/or probes useful for the specific detectionof MREJ types i, ii, iii, iv, v and vii are provided. For example, insome embodiments, primers and/or probes that hybridize under stringentconditions to each of the following SEQ ID NOs or the complementsthereof are provided: SEQ ID NOs: 99, 199, 144, 150, 155, and 163, suchas primers and/or probes that comprise, consist essentially of, orconsist of at least one of the following SEQ ID NOs: 99, 199, 144, 150,155, and 163. Optionally, at least one probe comprising anoligonucleotide that hybridizes under stringent conditions to SEQ IDNOs: 126, 128, 130 and 131 or the complement thereof is provided, forthe detection of MREJ sequences of types i, ii, iii, iv, v and vii. Forexample, at least one primer and/or probe that comprises, consistsessentially of, or consists of at least one of the following SEQ ID NOs:126, 128, 130 and 131, is provided.

In other preferred embodiments, the at least one primer(s) and/orprobe(s) that anneal to MREJ sequences comprises a pair ofoligonucleotides that hybridize under stringent conditions to SEQ IDNOs: 99 and 199 (for the detection of type i and type ii MREJ); SEQ IDNOs: 99 and 144 (for the detection of type iii MREJ); SEQ ID NOs: 99 and150 (for the detection of type iv MREJ); SEQ ID NOs: 99 and 155 (for thedetection of type v MREJ); and SEQ ID NOs: 99 and 163 (for the detectionof type vii MREJ), or the complement thereof. Optionallyoligonucleotides that hybridize under stringent conditions to each ofSEQ ID NOs: 99 and 199 (for the detection of type i and type ii MREJ);SEQ ID NOs: 99 and 144 (for the detection of type iii MREJ); SEQ ID NOs:99 and 150 (for the detection of type iv MREJ); SEQ ID NOs: 99 and 155(for the detection of type v MREJ); and SEQ ID NOs: 99 and 163 (for thedetection of type vii MREJ) are provided. Optionally, the sample is alsocontacted with a probe comprising an oligonucleotide that hybridizesunder stringent conditions to the nucleic acid of SEQ ID NOs: 9, 10, 11,12, 126, 128, 130 or 131, for the detection of MREJ sequences, or SEQ IDNOs: 9, 10, 11, and 12 for the detection of S. aureus nuc, or thecomplements thereof.

In preferred embodiments, the nuc-specific primer(s) and/or probe(s)comprise at least one first primer pair that hybridizes under stringentconditions to the following oligonucleotide pairs or the complementsthereof: SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5,SEQ ID NOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ IDNOs: 4 and 7; or SEQ ID NOs: 4 and 8; for the detection of S. aureus ina sample. For example, in some embodiments, the nuc-specific primer(s)and/or probe(s) comprise at least one first primer pair that comprises,consists essentially of, or consists of: SEQ ID NOs: 1 and 5; SEQ IDNOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ ID NOs: 3and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4 and 8.Optionally, in embodiments where the sample is contacted with a firstprimer pair comprising oligonucleotides that hybridize under stringentconditions to SEQ ID NOs: 1 and 5, or 1 and 6, or the complementsthereof, (e.g., oligonucleotides that comprise, consist essentially of,or consist of SEQ ID NOs: 1 and 5, or SEQ ID NOs: 1 and 6), the samplecan also be contacted with a probe comprising an oligonucleotide thathybridizes under stringent conditions to SEQ ID NO: 9 (e.g., SEQ ID NO:10) or the complement thereof. Optionally, in embodiments where thesample is contacted with a first primer pair comprising oligonucleotidesthat hybridize under stringent conditions to SEQ ID NOs: 3 and 7, or SEQID NOs: 3 and 8, or the complements thereof, (e.g., oligonucleotidesthat comprise, consist essentially of, or consist of SEQ ID NOs: 3 and7, or SEQ ID NOs: 3 and 8), the sample can also be contacted with aprobe comprising an oligonucleotide that hybridizes under stringentconditions SEQ ID NO: 11 (e.g., SEQ ID NO: 12) or the complementthereof. Preferably, the first primer pair comprises oligonucleotidesthat hybridize under stringent conditions to SEQ ID NOs: 1 and 6; or SEQID NOs: 3 and 8 or the complements thereof.

Optionally, the sample is also contacted with at least one probecomprising an oligonucleotide that hybridizes under stringent conditionsto SEQ ID NOs: 9, 10, 11, 12, for the detection of S. aureus nucsequences, or to SEQ ID NOs: 126, 128, 130 or 131, for the detection ofMREJ sequences, or the complement thereof, e.g., at least one probe thatcomprises, consists essentially of, or consists of SEQ ID NOs: 9, 10,11, 12, 126, 128, 130 or 131.

The presence and/or amount of annealed probe(s) can be detected, or theamount of an amplification product produced through annealing of theprimers to the nucleic acids can be detected, as an indication of thepresence and/or amount of S. aureus, and as an indication of thepresence and/or amount of MRSA.

Compositions and Kits

Provided herein are also compositions and kits that comprise, consistessentially of, or consist of oligonucleotides described herein.Preferably, oligonucleotides are between 10 and 45 nucleotides inlength. For example, oligonucleotides can be at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35or more nucleotides in length. As will be understood by those skilled inthe art, the nucleic acids of the embodiments disclosed herein can besingle-stranded (coding or antisense), or double-stranded, and may be aDNA (genomic, cDNA, or synthetic) or RNA molecule. Additional coding ornon-coding sequences may, but need not, be present within a nucleic acidof the embodiments disclosed herein, and a nucleic acid may, but neednot, be linked to other molecules and/or support materials.

Accordingly, some embodiments comprise, consist essentially of, orconsist of, at least one oligonucleotide of between about 10 to about 45nucleotides, and preferably at least 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35 nucleotides in lengthwhich hybridizes under stringent conditions with any of nucleic acids ofthe following sequences derived from S. aureus nuc sequences or thecomplements thereof: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or12, for example, oligonucleotides that comprise, consist essentially of,or consist of at least one of the following SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12. Preferred embodiments comprise, consistessentially of, or consist of a primer pair that hybridizes understringent conditions with any of the pairs of the following SEQ ID NOs:1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6;SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ IDNOs: 4 and 8, or the complements thereof, for example primer pairs thatcomprise, consist essentially of, or consist of the following SEQ IDNOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; orSEQ ID NOs: 4 and 8. In some embodiments, at least one probe comprisingan oligonucleotide that hybridizes under stringent conditions SEQ IDNOs: 9 and 11 (e.g., SEQ ID NOs: 10 and 12), or the complement thereof,is provided.

Other aspects relate to compositions useful for the detection of S.aureus and MRSA in a single reaction. Accordingly, some embodimentscomprise, consist essentially of, or consist of, at least one primerand/or probe that is preferably between about 10 to about 45 nucleotidesin length, such as an oligonucleotide that is at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35in length that hybridizes to an S. aureus-specific nuc sequence, and atleast one primer and/or probe that hybridizes to at least one MREJsequence of MREJ types i-xx. Some embodiments provide at least twoprimer pairs, wherein a first primer pair hybridizes under stringentconditions to S. aureus-specific nuc sequences (e.g., SEQ ID NOs: 1 and5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6; SEQ IDNOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ ID NOs: 4and 8) and a second primer pair hybridizes to MREJ sequences (e.g., SEQID NOs: 92 and 82; 92 and 83; 92 and 84; 104 and 86; 104 and 87; 104 and88; 99 and 89; 99 and 199 (for the detection of MREJ type i); SEQ IDNOs: 92 and 82; 92 and 129; 92 and 130; 93 and 83; and 92 and 84; 99 and89; 99 and 199 (for the detection of MREJ type ii); SEQ ID NOs: 92 and136; 92 and 137; 92 and 138; 99 and 202; 99 and 144 (for the detectionof MREJ type iii); SEQ ID NOs: 92 and 141; 99 and 105; 99 and 150 (forthe detection of MREJ type iv); SEQ ID NOs: 92 and 146; 99 and 196; 99and 155 (for the detection of MREJ type v); SEQ ID NOs: 92 and 152; 99and 161; (for the detection of MREJ type vi); SEQ ID NOs: 92 and 153; 92and 154; 99 and 162; 99 and 163 (for the detection of MREJ type vii);SEQ ID NOs: 92 and 162; 92 and 163; 99 and 170 (for the detection ofMREJ type viii); SEQ ID NOs: 92 and 168; 99 and 177 (for the detectionof MREJ type ix); SEQ ID NOs: 197 and an oligonucleotide that hybridizesunder stringent conditions to orf22 (for the detection of MREJ type x);SEQ ID NOs: 189 and 106; 189 and 99; 189 and 190; 189 and 109 (for thedetection of MREJ type xi); SEQ ID NOs: 194 and 106; 194 and 99; 104 and191; 194 and 109 (for the detection of MREJ type xii); SEQ ID NOs: 177and 106; 177 and 99; 177 and 190; and 177 and 109 (for the detection ofMREJ type xiii); SEQ ID NOs: 177 and 106; 177 and 99; 177 and 193; 177and 109 (for the detection of MREJ type xiv); SEQ ID NOs: 184 and 106;108 and 99; 184 and 191; 184 and 191 (for the detection of MREJ typexv); SEQ ID NOs: 89 and 109 (for the detection of MREJ type xvi); SEQ IDNOs: 185 and 106; 185 and 99; 185 and 191; 185 and 109 (for thedetection of MREJ type xvii); SEQ ID NOs: 186 and 106; 186 and 99; 186and 193; 186 and 109 (for the detection of MREJ type xviii); SEQ ID NOs:187 and 106; 107 and 99; 187 and 913; 187 and 109 (for the detection ofMREJ type xix); SEQ ID NOs: 188 and 106; 188 and 99; 188 and 913; and188 and 109 (for the detection of MREJ type xx)). In some embodiments,at least one probe(s) that can hybridize to amplification productsproduced by an S. aureus-specific nuc primer pair and/or MREJ-specificprimer pair described herein is also provided (e.g., SEQ ID NOs: 9, 10,11, 12, 126, 128, 130 or 131).

Accordingly, some embodiments comprise, consist essentially of, orconsist of primer pairs that hybridize under stringent conditions to thenucleic acid sequences of:

SEQ ID NOs: 1 and 6

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163, or the complements thereof.

Other embodiments comprise, consist essentially of, or consist of aplurality of primer pairs, wherein the primers anneal under stringentconditions to the nucleic acid sequences of:

SEQ ID NOs: 3 and 8;

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163, or the complements thereof.

Still other aspects relate to kits for the detection and/orquantification of S. aureus, or S. aureus and MRSA. In some embodiments,the kits comprise, consist essentially of, or consist of, at least oneoligonucleotide of between about 10 to about 45 nucleotides in length,for example at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 31, 32, 33, 34, 35 nucleotides in length, whichhybridizes under stringent conditions with any of nucleic acids of thefollowing sequences derived from S. aureus nuc sequences or thecomplements thereof: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or12. Preferred embodiments provide kits that comprise, consistessentially of, or consist of a primer pair that hybridizes understringent conditions with any of the pairs of the following SEQ ID NOs:1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ ID NOs: 2 and 6;SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4 and 7; or SEQ IDNOs: 4 and 8, or the complements thereof. In some embodiments, the kitprovides at least one probe comprising an oligonucleotide thathybridizes under stringent conditions SEQ ID NOs: 9 and 11 (e.g., SEQ IDNOs: 10 and 12), or the complement thereof.

Other embodiments provide kits useful for the detection of S. aureus andMRSA together. In some embodiments, the kits comprise, consistessentially of, or consist of, at least one primer and/or probe that isbetween about 10 to about 45 nucleotides in length, for example, atleast 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 31, 32, 33, 34, 35 nucleotides in length, that hybridizes to an S.aureus-specific nuc sequence, and at least one primer and/or probe thathybridizes to at least one MREJ sequence of MREJ types i-xx. Someembodiments provide kits, wherein the kits include at least two primerpairs. A first primer pair can hybridize under stringent conditions toS. aureus-specific nuc sequences (e.g., primers that are at least 10nucleotides in length and can hybridize under stringent conditions toSEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 5, SEQ IDNOs: 2 and 6; SEQ ID NOs: 3 and 7; SEQ ID NOs: 3 and 8; SEQ ID NOs: 4and 7; or SEQ ID NOs: 4 and 8, or the complement thereof) and a secondprimer pair can hybridize under stringent conditions to MREJ sequences(e.g., primers that are at least 10 nucleotides in length and canhybridize under stringent conditions to SEQ ID NOs: 92 and 82; 92 and83; 92 and 84; 104 and 86; 104 and 87; 104 and 88; 99 and 89; 99 and 199(for the detection of MREJ type i); SEQ ID NOs: 92 and 82; 92 and 129;92 and 130; 93 and 83; and 92 and 84; 99 and 89; 99 and 199 (for thedetection of MREJ type ii); SEQ ID NOs: 92 and 136; 92 and 137; 92 and138; 99 and 202; 99 and 144 (for the detection of MREJ type iii); SEQ IDNOs: 92 and 141; 99 and 105; 99 and 150 (for the detection of MREJ typeiv); SEQ ID NOs: 92 and 146; 99 and 196; 99 and 155 (for the detectionof MREJ type v); SEQ ID NOs: 92 and 152; 99 and 161; (for the detectionof MREJ type vi); SEQ ID NOs: 92 and 153; 92 and 154; 99 and 162; 99 and163 (for the detection of MREJ type vii); SEQ ID NOs: 92 and 162; 92 and163; 99 and 170 (for the detection of MREJ type viii); SEQ ID NOs: 92and 168; 99 and 177 (for the detection of MREJ type ix); SEQ ID NOs: 197and an oligonucleotide that hybridizes under stringent conditions toorf22 (for the detection of MREJ type x); SEQ ID NOs: 189 and 106; 189and 99; 189 and 190; 189 and 109 (for the detection of MREJ type xi);SEQ ID NOs: 194 and 106; 194 and 99; 104 and 191; 194 and 109 (for thedetection of MREJ type xii); SEQ ID NOs: 177 and 106; 177 and 99; 177and 190; and 177 and 109 (for the detection of MREJ type xiii); SEQ IDNOs: 177 and 106; 177 and 99; 177 and 193; 177 and 109 (for thedetection of MREJ type xiv); SEQ ID NOs: 184 and 106; 108 and 99; 184and 191; 184 and 191 (for the detection of MREJ type xv); SEQ ID NOs: 89and 109 (for the detection of MREJ type xvi); SEQ ID NOs: 185 and 106;185 and 99; 185 and 191; 185 and 109 (for the detection of MREJ typexvii); SEQ ID NOs: 186 and 106; 186 and 99; 186 and 193; 186 and 109(for the detection of MREJ type xviii); SEQ ID NOs: 187 and 106; 107 and99; 187 and 913; 187 and 109 (for the detection of MREJ type xix); SEQID NOs: 188 and 106; 188 and 99; 188 and 913; and 188 and 109 (for thedetection of MREJ type xx) or the complements thereof). In someembodiments, the kits include at least one probe(s) that can hybridizeunder stringent conditions to amplification products produced by an S.aureus-specific nuc primer pair and/or MREJ-specific primer pairdescribed herein is also provided (e.g., a probe comprising anoligonucleotide that can hybridize under stringent conditions to SEQ IDNOs: 9, 10, 11, 12, 126, 128, 130 or 131 or the complement thereof).

Accordingly, some embodiments provide kits that comprise, consistessentially of, or consist of primer pairs that hybridize understringent conditions to the nucleic acid sequences of:

SEQ ID NOs: 1 and 6

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163, or the complements thereof.

Other embodiments provide kits that comprise, consist essentially of, orconsist of a plurality of primer pairs, wherein the primers anneal understringent conditions to the nucleic acid sequences of:

SEQ ID NOs: 3 and 8;

SEQ ID NOs: 99 and 199;

SEQ ID NOs: 99 and 144;

SEQ ID NOs: 99 and 150;

SEQ ID NOs: 99 and 155; and

SEQ ID NOs: 99 and 163, or the complements thereof.

The diagnostic kits, primers and probes disclosed herein can be used todetect and/or identify S. aureus, as well as detect and/or identify bothS. aureus and MRSA of MREJ types i to xx, in both in vitro and/or insitu applications. For example, it is contemplated that the kits may beused in combination with any previously described primers/probes fordetecting MRSA of MREJ types i to xx. It is also contemplated that thediagnostic kits, primers and probes disclosed herein can be used aloneor in combination with any other assay suitable to detect and/oridentify microorganisms, including but not limited to: any assay basedon nucleic acids detection, any immunoassay, any enzymatic assay, anybiochemical assay, any lysotypic assay, any serological assay, anydifferential culture medium, any enrichment culture medium, anyselective culture medium, any specific assay medium, any identificationculture medium, any enumeration culture medium, any cellular stain, anyculture on specific cell lines, and any infectivity assay on animals.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting.

EXAMPLE 1

This example illustrates the utility of various primer pairs, chosen foroptimized, specific detection of S. aureus from a sample using PCR. SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8 were designed to anneal to S.aureus-specific regions of the nuc gene. PCR reaction mixtures included0.5 μM of each of the indicated primers, 0.2 mM dNTPs (Roche), 2 mMMgCl₂ (SIGMA), 1 unit FASTSTART™ Taq DNA polymerase (Roche), 50 mM Tris(EMD), 10 mM KCl (Laboratoire Mat), and 5 mM (NH₄)₂SO₄ (SIGMA).

For each primer pair tested, three replicates containing varying amountsof chromosomal template DNA were run. One set of reactions included 15copies of chromosomal template DNA from S. aureus strain ATCC 43300(MRSA). Another set of reactions included 185 copies of ATCC 43300template DNA. A negative control was also run, which did not have anyadded template DNA. Parallel sets of reactions were run with chromosomaltemplate DNA from S. aureus strain ATCC 25923 (MSSA).

PCR reactions were performed using a SMARTCYCLER® QT-PCR machine(Cepheid). The cycling parameters were as follows: 95° C. for 900 min,followed by 45 cycles of 95° C. for 5 sec, 59° C. for 15 sec and 72° C.for 20 sec. Amplified products were visualized on agarose gels (FIGS. 1Aand 1B).

As shown in FIGS. 1A and 1B, the following primer pairs showedparticularly good results in the specific amplification of DNA from bothMSSA and MRSA S. aureus strains:

SEQ ID NOs: 1 and 5

SEQ ID NOs: 1 and 6

SEQ ID NOs: 2 and 6

SEQ ID NOs: 4 and 7

SEQ ID NOs: 4 and 8

SEQ ID NOs: 3 and 7, and

SEQ ID NOs: 3 and 8.

The primer pair SEQ ID NOs: 2 and 5 was less sensitive, as indicated bythe relative amount of amplification product produced, compared to otherprimer pairs.

EXAMPLE 2

The ability to detect S. aureus and to identify MRSA in a singlereaction was tested. A multiplex PCR reaction was designed to includeprimers that anneal under standard PCR conditions to the S. aureusspecies-specific orfX sequence and a sequence of SCCmec right extremityjunction (MREJ) of the most commonly clinically encountered MRSA types(i.e., MRSA of MREJ types i, ii, iii, iv, v, vii). SEQ ID NOs: 99, 199,144, 150, 155 and 163 were used for the detection of MRSA of MREJ typesi, ii, iii, iv, v, and vii. Primers that anneal to S. aureus specificregions of the nuc gene under the same conditions (SEQ ID NOs: 3 and 8)were used in the reaction for the detection of both MRSA and MSSAstrains in the test reactions. Molecular beacon probes which aredetectable on the SMARTCYCLER® apparatus at FAM, Texas Red and TETchannels were designed for hybridization to amplification products ofthe MRSA specific reactions (SEQ ID NOs: 126 and 130), nuc/S. aureusspecific reactions (SEQ ID NO: 12), and an internal control,respectively.

PCR reactions included 0.9 μM SEQ ID NO: 99, 0.4 μM SEQ ID NO: 199; 0.6μM SEQ ID NO: 144, 0.3 μM SEQ ID NO: 150, 0.2 μM SEQ ID NO: 155, 0.7 μMSEQ ID NO: 163, 0.1 μM SEQ ID NO: 3, 0.1 μM SEQ ID NO: 8, 0.1 μM SEQ IDNO: 126, 0.1 μM SEQ ID NO: 130, 0.25 μM SEQ ID NO: 12, 0.2 μM controlDNA, 0.3 mM dNTPs (Roche) 4 mM MgCl₂ (SIGMA), 2.8 units FASTSTART® TaqDNA polymerase (Roche), 100 mM Tris, pH 8.3 (EMD), 10 mM KCl(Laboratoire Mat), 5 mM (NH4)₂SO₄ (SIGMA), 0.15 mg/mL BSA (SIGMA), 4%Trehalose (SIGMA), 3000 copies of internal control template DNA, 2780copies of S. epidermidis chromosomal DNA, and either 0, 2.5, 5, 10, 15or 20 copies MSSA chromosomal DNA (isolated from ATCC strain 25923) or0, 2.5, 5, 10 or 20 copies of MRSA chromosomal DNA (isolated from ATCCstrain 43300).

PCR reactions were performed in a SMARTCYCLER® instrument (Cepheid).Cycling parameters were as follows: 95° C. for 900 min, followed by 45cycles of 95° C. for 5 sec, 59° C. for 15 sec and 72° C. for 20 sec. Thefluorescence was continuously measured at the appropriate wavelengths,and is graphically depicted in FIGS. 2A and 2B.

FIG. 2A depicts the fluorescence readings of reactions containing MSSAtemplate DNA. Under these reaction conditions, 2.5 copies of MSSA DNAwere easily detected (Texas Red Channel), demonstrating the utility ofSEQ ID NOs: 3, 8 and 12 in multiplex PCR. As expected, positive signalsare also present in the TET channel indicating that the internal controlworked properly, and that no inhibitors were present in the reactions.

FIG. 2B depicts the fluorescence readings of reactions containing MRSAtemplate DNA. Under these reaction conditions, 2.5 copies of MRSA DNAwere easily detected (FAM channel). This demonstrates the utility of SEQID NOs: 99, 199, 150, 155, 144, 126, and 130 in a multiplex PCR that candetect all S. aureus strains, including MRSA. As shown in the Texas-Redchannel, the nuc-specific primers and probes (SEQ ID NOs: 3, 8, and 12)detected 2.5 copies of DNA. Positive signals are also present in the TETchannel, indicating that the internal control worked properly, and thatno inhibitors were present in the reactions.

This example highlights the very high sensitivity obtainable with a PCRmultiplex assay that amplifies MREJ sequences from MRSA and nucsequences from S. aureus with an internal control.

EXAMPLE 3

The specificity of a multiplex PCR assay that amplifies MREJ sequencesfrom MRSA and the nuc sequence from S. aureus was analyzed. ChromosomalDNA from 80 bacterial species other than S. aureus was used as templateDNA in a multiplex PCR assay as described in Example 2. The strainstested are enumerated in Table 2.

1 ng of chromosomal DNA isolated from each species indicated in Table 1was used in a separate reaction containing 0.9 μM SEQ ID NO: 99, 0.4 μMSEQ ID NO: 199; 0.6 μM SEQ ID NO: 144, 0.3 μM SEQ ID NO: 150, 0.2 μM SEQID NO: 155, 0.7 μM SEQ ID NO: 163, 0.1 μM SEQ ID NO: 3, 0.1 μM SEQ IDNO: 8, 0.1 μM SEQ ID NO: 126, 0.1 μM SEQ ID NO: 131, 0.25 μM SEQ ID NO:11, 0.2 μM internal control DNA, 0.3 mM dNTPs (Roche) 4 mM MgCl₂(SIGMA), 2.8 units FASTSTART® Taq DNA polymerase (Roche), 100 mM Tris,pH 8.3 (EMD), 10 mM KCl (Laboratoire Mat), 5 mM (NH₄)₂SO₄ (SIGMA), 0.15mg/mL BSA (SIGMA), 4% Trehalose (SIGMA), 3000 copies of internal controltemplate DNA, and 2780 copies of S. epidermidis DNA.

Each reaction was performed in triplicate. The reactions were allowed toproceed following the parameters set forth in Example 2. Table 2summarizes the results of the reactions. No positive signal was observedin the FAM and Texas Red channels for the 80 different species tested. Apositive signal was detected in the TET channel for each of the 80different species tested, indicating that the reactions did not containinhibitors. The algorithm of interpretation of results is summarized inTable 3.

TABLE 2 PCR results S. aureus Strain MRSA IC (Texas Species number (FAM)(TFT) Rad) Acinetobacter baumannii ATCC 19606 − + − Acinetobacter IwoffiCDCF 3697 − + − Actinomyces israelii ATCC 12102 − + − Actinomycespyogenes ATCC 19411 − + − Bacillus cereus ATCC 14579 − + − Bacteroidesfragilis ATCC 25285 − + − Bifidobacterium breve ATCC 15700 − + −Bordetella pertusis ATCC 9797 − + − Corynebacterium genitalium LSPQ3583− + − Corynebacterium aquaticus ATCC 14665 − + − Corynebacterium bovisATCC 7715 − + − Corynebacterium flavescens ATCC 10340 − + − Enterobactercloacae ATCC 13047 − + − Enterococcus faecalis ATCC19433 − + −Enterococcus faecium ATCC 19434 − + − Enterococcus flavescens ATCC 49996− + − Enterrococcus gallinarum ATCC 49573 − + − Enterrococcus hirae ATCC8043 − + − Escherichia coli ATCC 23511 − + − Helicobacter pyloriIDI-2019 − + − Fusobacterium nucleatum subsp. ATCC 10953 − + −Polymorphum Gardnerella vaginalis ATCC 14019 − + − Haemophilusinfluenzae ATCC 9006 − + − Homo sapiens 2.16 − + − Klebsiella pneumoniaeATCC 13883 − + − Lactobacillus crispatus ATCC 33820 − + − Listeriamonocytogenes L 374 − + − Micrococcus luteus ATCC 9341 − + − Moraxellacatarrhalis ATCC 43628 − + − Neisseria gonorrhoeae ATCC 35201 − + −Neisseria meningitides ATCC 13077 − + − Pasteurella aerogenes ATCC 27883− + − Peptostreptococcus anaerobius ATCC 27337 − + − PeptostreptococcusLSPQ 2639 − + − asaccharolyticus Porphyromonas asaccharolytica ATCC25260 − + − Prevotella melaninogenica ATCC 25845 − + − Propionibacteriumacnes ATCC 6919 − + − Proteus mirabilis ATCC 29906 − + − Pseudomonasaeruginosa ATCC 35554 − + − Pseudomonas fluorescens ATCC 13525 − + −Salmonella typhimurium ATCC 14028 − + − Serratia marcescens ATCC 13880− + − Shigella sonnei ATCC 29930 − + − Staphylococcus arlettae CCRI-9265− + − Staphylococcus auricularis R413 − + − Staphylococcus capitisCCRI-9572 − + − Staphylococcus caprae CCRI-9117 − + − Staphylococcuscamosus R714 − + − Staphylococcus chromogenes ATCC 43764 − + −Staphylococcus cohnii subsp. R570 − + − Urealyticum Staphylococcusdelphini ATCC 49171 − + − Staphylococcus epidermidis ATCC 35984 − + −Staphylococcus equorum ATCC 43958 − + − Staphylococcus fells ATCC 49168− + − Staphylococcus gallinarum ATCC 35539 − + − Staphylococcushaemolyticus ATCC 29970 − + − Staphylococcus hominis CCRI-1347 − + −Staphylococcus intermedius ATCC 29663 − + − Staphylococcus kloosii ATCC43959 − + − Staphylococcus lentus ATCC 29070 − + − Staphylococcuslugdunensis ATCC 43809 − + − Staphylococcus pasteuri ATCC 51129 − + −Staphylococcus pulvereri ATCC 51698 − + − Staphylococcus saprophyticusATCC 15305 − + − Staphylococcus sciuri R573 − + − Staphylococcussimulans ATCC 27848 − + − Staphylococcus warneri ATCC 35985 − + −Staphylococcus xylosus LSPQ2517 − + − Streptococcus agalactiae ATCC12973 − + − Streptococcus anginosus ATCC 33397 − + − Streptococcus mitisATCC 49456 − + − Streptococcus mutans ATCC 25175 − + − Streptococcuspneumoniae ATCC 49619 − + − Streptococcus pyogenes ATCC 12384 − + −Streptococcus salivarius ATCC 7073 − + − Streptococcus sanguinis ATCC10556 − + − Streptococcus suis ATCC 43765 − + − Yersinia enterocoliticaATCC 23715 − + − Candida albicans ATCC 10231 − + − Candida qlabrata ATCC66032 − + −

TABLE 3 FAM Assay Texas-Red IC (TET) Result Assay Result Result ReportedReported Reported Interpretation of Result Negative Negative PASS No S.aureus DNA detected Positive Positive or N/A MRSA DNA detected NegativeNegative Positive N/A S. aureus DNA detected, no MRSA DNA detectedUnresolved Fail Unresolved- inhibitory specimen or reagent failure

This example highlights the complete specificity reached with a PCRmultiplex assay that amplifies MREJ sequences from MRSA and nuc sequencefrom S. aureus with an internal control.

EXAMPLE 4

The ability of a multiplex PCR assay that amplifies MREJ sequences fromMRSA and nuc sequence from S. aureus to accurately detect S. aureus andidentify MRSA directly from wound specimens was tested.

A multiplex PCR reaction was designed to include primers to amplifysequences specific to the MREJ regions of the most clinically relevantMRSA (e.g., primers that anneal to S. aureus species-specific orfXsequences and SCCmec sequences), as well as primers that anneal to S.aureus specific regions of the nuc gene of all S. aureus strains (e.g.,MRSA and MSSA), under the same conditions. Briefly, SEQ ID NOs: 99, 199,144, 150, 155 and 163 were used for amplification of sequences of theMREJ region of various MRSA of MREJ types i, ii, iii, iv, v, and vii.Primers that anneal to S. aureus specific regions of the nuc gene underthe same conditions (SEQ ID NOs: 3 and 8) were used in the reaction forthe detection of both MRSA and MSSA strains in the test reactions.Molecular beacon probes which are detectable on the SMARTCYCLER®apparatus at FAM, Texas Red and Tet channels were designed forhybridization to amplification products of the MRSA specific reactions(SEQ ID NOs: 126 and 130), nuc/S. aureus specific reactions (SEQ ID NO:12), and the internal control, respectively.

One hundred and three wound samples were collected on patients usingAmies liquid swabs (Copan Diagnostics, Inc). Samples were cultured andsubcultured on blood agar plates (Becton Dickinson). Based on theirmorphology, suspected S. aureus were identified with a coagulase test(Jorgenson, J. H., and W. E. Kloos. 1987. Staphylococcal Infections, inB. B. Wentworth (ed.), Diagnostic procedures for bacterial infections,7th ed., American Public Health Association, Washington, D. C.) and insome cases with latex agglutination (Staphaurex, Remel Inc.) Methicillinresistance was determined using the VITEK™ bacterial identificationsystem (bioMérieux, Durham, N.C.).

DNA was isolated from the isolates using the IDI™ lysis kit (GeneOhmSciences, Inc.). A swab of the isolate was broken in 1 mL of TE buffer(10 mM Tris, 1 mM EDTA, pH 8.0) and vortexed for 1 min at high speed. 50μL of the cell suspensions were transferred to a lysis tube containingglass beads and vortexed for 5 minutes at high speed. The tubes werecentrifuged at 13,000 rpms for 2 min and heated at 95° C. for 2 minutes.The tube was placed on ice until used in the reaction.

3 μL of the lysis reaction was added to a PCR mix that contained 0.9 μMSEQ ID NO: 99, 0.4 μM SEQ ID NO: 199, 0.6 μM SEQ ID NO: 144, 0.3 μM SEQID NO: 150, 0.2 μM SEQ ID NO: 155, 0.7 μM SEQ ID NO: 163, 0.1 μM SEQ IDNO: 3, 0.1 μM SEQ ID NO: 8, 0.1 μM SEQ ID NO: 126, 0.1 μM SEQ ID NO:130, 0.25 μM SEQ ID NO:12, 0.2 μM internal control DNA, 0.3 μM dNTPs(Roche), 4 mM MgCl₂ (SIGMA), 2.8 units FASTSTART® Taq polymerase(Roche), 100 mM Tris, pH 8.3 (EMD), 10 mM KCl (LaboratoireMat), 5 mM(NH₄)₂SO₄ (SIGMA), 0.15 mg/mL BSA (SIGMA) 4% trehalose (SIGMA), 3000copies internal control DNA, and 2780 copies S. epidermidis chromosomalDNA.

PCR was carried out in a SMARTCYCLER® (Cepheid) using the same cyclingparameters as described in Example 2. For each specimen, the cyclethreshold (CT) in FAM, Texas-Red, and TET channels was determined usingthe SMARTCYCLER® software. Assay results were interpreted as indicatedin Table 3:

The multiplex PCR assay above is designed such that any S. aureus strainproduces a positive signal in the Texas-Red channel. The presence of aclinically relevant MRSA will produce a positive signal in the FAMchannel. Accordingly, a negative result in the FAM channel combined witha positive result in the Texas-Red channel is indicative of the presenceof MSSA.

In instances where a discordant result appeared between culture assaysdescribed above, and the multiplex PCR reaction, Tryptic Soy Broth wasadded to the TE buffer tube containing the swab, and incubatedovernight, at 35° C. 50 μL of the overnight culture was plated on bloodagar plates and isolates were identified as MRSA, MSSA, or negative (noS. aureus).

The data collected are depicted in Tables 4A, 4B and 4C, below.

TABLE 4A (A) PCR MRSA MSSA Negative Total Unresolved Cul- MRSA  27 (32)*0 (0) 0 (0) 27 (32) 1 (0) ture MSSA 2 (2) 18 (19) 1 (0) 21 (21) 1 (0)Nega- 4 (0) 1 (1) 43 (45) 48 (46) 5 (4) tive Total 33 (34) 19 (20) 44(45) 96 (99) 7 (4) *before resolution of discordant results (afterresolution of discordant results)

TABLE 4B before resolution after resolution MRSA sensitivity  100%(27/27)  100% (32/32) MSSA sensitivity 85.7% (18/21) 90.5% (19/21)S.aureus sensitivity 97.9% (47/48)  100% (53/53) Specificity 89.6%(43/48) 97.8% (45/46) Unresolved  6.8% (7/103)  3.9% (4/103)

TABLE 4C

As shown in Tables 4A and 4B, the multiplex assay is 100% sensitive forMRSA, indicating that every positive MRSA result achieved in the PCRassay corresponded to a positive result in the culture identification,both before and after resolution. The sensitivity of the PCR assay forS. aureus detection after resolution was 90.5%, with 19 of 21 of MSSAstrains showing a positive result in the PCR assay. Importantly,however, the two strains that were incorrectly identified as not beingMSSA are strains that were formerly MSSA but lost a portion of theSCCmec element and retained the junction near orfX to which the PCRamplification primers hybridize.

Table 4C shows the individual PCR and culture results for each of the103 wound specimens following the resolution of discordant results. Theshaded entries indicate that the results obtained in the culture testand in the PCR assay were in agreement. The column labeled (CT)indicates the PCR cycle in which a positive signal becomes detectableover the background noise. As shown in the table, four samples were notable to be resolved in the PCR assay, due to the presence of reactioninhibitors in the sample.

The results above demonstrate the high sensitivity and specificity ofthe multiplex PCR assay applied directly to wound specimens.Accordingly, the multiplex assay offers the first convenient, reliable,sensitive, and specific assay specific for both MRSA and MSSA.

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments, they are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

Numerous literature and patent references have been cited in the presentpatent application. Each and every reference that is cited in thispatent application is hereby expressly incorporated by reference in itsentirety.

What is claimed is:
 1. A method of specifically detecting the presenceof a Staphylococcus aureus (S. aureus) strain and identifying amethicillin-resistant S. aureus strain from a clinical sample in asingle assay, comprising contacting said sample with at least one primerand/or probe of at least 11 nucleotides that anneals under stringentconditions to an S. aureus-specific sequence within SEQ ID NO: 200 orthe complement thereof, or any sequence which differs from SEQ ID NO:200 by 1 to 20 nucleotides; contacting said sample with at least oneprimer and/or probe of at least 10 nucleotides that anneals understringent conditions to at least one of the following MREJ specific SEQID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, or 88 or the complement thereof; and detecting thepresence and/or amount of annealed probe(s), or detecting the amount ofan amplification product produced through annealing of the primers tothe nucleic acids, as an indication of the presence and/or amount of S.aureus, and as an indication of the presence and/or amount of MRSA,wherein said stringent conditions comprise 4 mM MgCl₂, 100 mM Tris (pH8.3), 10 mM KCl, 5 mM (NH₄)₂SO₄, 0.15 mg/mL BSA, 4% trehalose at 59° C.